Targeting MERTK on tumour cells and macrophages: a potential intervention for sporadic and NF2-related meningioma and schwannoma tumours

Foram Dave; Kevin Herrera; Alex Lockley; Laurien L van de Weijer; Summer Henderson; Agbolahan A Sofela; Laura Hook; Claire L Adams; Emanuela Ercolano; David A Hilton; Emmanuel A Maze; Kathreena M Kurian; Sylwia Ammoun; C Oliver Hanemann

doi:10.1038/s41388-024-03131-z

. 2024 Aug 23;43(41):3049–3061. doi: 10.1038/s41388-024-03131-z

Targeting MERTK on tumour cells and macrophages: a potential intervention for sporadic and NF2-related meningioma and schwannoma tumours

Foram Dave ^1,^#, Kevin Herrera ^1,^#, Alex Lockley ¹, Laurien L van de Weijer ¹, Summer Henderson ¹, Agbolahan A Sofela ¹, Laura Hook ¹, Claire L Adams ¹, Emanuela Ercolano ¹, David A Hilton ², Emmanuel A Maze ¹, Kathreena M Kurian ³, Sylwia Ammoun ^1,^✉,^#, C Oliver Hanemann ^1,^✉,^#

PMCID: PMC11458476 PMID: 39179860

Abstract

Meningioma and schwannoma are common tumours of the nervous system. They occur sporadically or as part of the hereditary NF2-related schwannomatosis syndrome. There is an unmet need for new effective drug treatments for both tumour types. In this paper, we demonstrate overexpression/activation of TAM (TYRO3/AXL/MERTK) receptors (TAMs) and overexpression/release of ligand GAS6 in patient-derived meningioma tumour cells and tissue. For the first time, we reveal the formation of MERTK/TYRO3 heterocomplexes in meningioma and schwannoma tissue. We demonstrate the dependence of AXL and TYRO3 expression on MERTK in both tumour types, as well as interdependency of MERTK and AXL expression in meningioma. We show that MERTK and AXL contribute to increased proliferation and survival of meningioma and schwannoma cells, which we inhibited in vitro using the MERTK/FLT3 inhibitor UNC2025 and the AXL inhibitor BGB324. UNC2025 was effective in both tumour types with superior efficacy over BGB324. Finally, we found that TAMs are expressed by tumour-associated macrophages in meningioma and schwannoma tumours and that UNC2025 strongly depleted macrophages in both tumour types.

graphic file with name 41388_2024_3131_Figa_HTML.jpg

Subject terms: CNS cancer, Targeted therapies

Introduction

Meningioma and schwannoma are common tumours of the nervous system and occur sporadically or as part of the hereditary syndrome NF2-related schwannomatosis (NF2, formerly Neurofibromatosis type-2) [1, 2]. NF2-related tumours, as well as 70% of sporadic schwannoma and 50–60% of sporadic meningioma have underlying Neurofibromin 2 (NF2) gene mutations leading to deficiency of the tumour suppressor Merlin [1, 3, 4].

While NF2-related/sporadic schwannoma and 80% of meningioma are usually benign, they can be associated with high morbidity especially when multiple or located near critical structures. [5]. Surgery/radiotherapy can be ineffective in patients with inoperable, aggressive, or recurrent tumours [6–8], hence the pressing need for effective drug therapies.

TYRO3, AXL and MERTK constitute the TAM family of receptor tyrosine kinases (TAMs), dysregulation of which is linked to neoplasms [9]. TAMs can be activated by growth arrest specific 6 (GAS6), protein S1 (PROS1), tubby (TUB), TUB like protein 1 (TULP1), or galectin3 (GAL3) ligands [10–12], contributing to tumour development/progression [9, 13, 14]. GAS6-induced activation of one of the TAM receptors requires the presence of another TAM receptor [15]. TAMs can also be activated in a ligand-independent manner via interaction with each other or through interactions with other receptor types on neighbouring cells [16, 17].

TAMs are also expressed by macrophages in tumours [18–20] promoting immune evasion [21]. Macrophages are present in meningioma and schwannoma tissues and NF2 mutations are linked to increased macrophages [22]. Meningioma/schwannoma macrophage counts are proportional to tumour aggressiveness/poor outcome [23–25]. We previously showed that AXL/GAS6 are involved in increased schwannoma cell proliferation/survival in vitro [26], and demonstrated increased meningioma (tissue) staining for pAXL/AXL/MERTK/GAS6 compared to normal meningeal tissue (NMT) [27]. Using patient-derived primary cells and tissue, we report that the overexpression of activated MERTK/AXL/TYRO3 is similar in low-grade (grade-I) and high-grades (grades-II/III) meningioma, independent of Merlin status. Additionally, we demonstrate that MERTK/AXL/TYRO3 are also expressed by tumour-associated macrophages and microglia in both tumour types. Using MERTK/AXL/TYRO3- shRNA, and MERTK/FLT3 and AXL inhibitors (UNC2025 and BGB324 respectively), we show that MERTK and AXL, but not TYRO3, are involved in increased proliferation/survival of meningioma and schwannoma cells. Finally, we demonstrate that MERTK is a superior therapeutic target over AXL in both tumour types and suggest targeting MERTK in tumour cells as well as tumour-associated macrophages as novel therapies.

Results

Expression of TAMs and GAS6 in meningioma and schwannoma

pMERTK/MERTK/pAXL/AXL/TYRO3 are strongly overexpressed in all meningioma grades compared to normal meningeal tissue (NMT). Only pTYRO3 overexpression was not significant (Fig. 1A, B and Supplementary Fig. 1A–L). GAS6 is overexpressed in grade-I meningioma tissue (MN-GI) (Fig. 1A and Supplementary Fig. 1C, H, I, K, L). RNA-seq demonstrated expression of GAS6, as well as other ligands PROS1, GAL3 and TULP1 in MN-GI tissue and cells (Supplementary Fig. 1O).

Fig. 1 — **A, B** MERTK, AXL and TYRO3 (A) and pMERTK and pAXL (B) expression is significantly increased in grade-I (MN-GI) and grades- II/III (MN-GII/III) meningioma tissue compared to normal meningeal tissue (NMT), with no significant difference between grades. Expression of pTYRO3 is increased in all meningioma grades compared to NMT but this result is not significant. C, D Adenovirus-mediated reintroduction of wild-type Merlin into Merlin-negative MN-GI primary cells resulted in a significant partial decrease in the expression of AXL, TYRO3 and MERTK, with no significant effect on GAS6. Western blot data were normalised to the loading control GAPDH. P values were calculated by Kruskal Wallis test followed by post hoc Dunn’s test (A, B) and ANOVA with Dunnett’s multiple comparison test (D) and are represented as follows: ns (not significant) P > 0.05, * P < 0.05, ** P < 0.01 and *** P < 0.001. Mean ± SEM is indicated in all graphs.

There is no significant difference in the expression of pMERTK/MERTK/pAXL/AXL/TYRO3/GAS6 between Merlin-positive and Merlin-negative MN-GI and MN-GII/III tissue (Supplementary Fig. 1A–N). Only pTYRO3 expression was higher in Merlin-positive compared to Merlin-negative MN-GI tissue (Supplementary Fig. 1M). There is no significant difference in the expression of these proteins between Merlin-positive and Merlin-negative cells (>p3, passage 3) (Supplementary Fig. 1P–T). Release of GAS6 was similar in both Merlin-positive and Merlin-negative MN-GI cells (>p3) (Supplementary Fig. 1U). Merlin reintroduction into Merlin-negative MN-GI cells (>p3) partially decreased the expression of MERTK/AXL/TYRO3, but not GAS6 (Fig. 1C, D).

Western blotting demonstrated increased expression of pAXL/AXL/pMERTK/MERTK/pTYRO3, and similar expression of TYRO3 in schwannoma tissue compared to normal nerve tissue (NNT) (Supplementary Fig. 1V, W, X). MERTK is glycosylated in schwannoma tissue (Supplementary Fig. 1Y).

A significant correlation between increased MERTK (p = 0.0096)/AXL (p = 0.0079)/TYRO3 (p = 0.04438)/GAS6 (p = 0.008) mRNA expression and decreased disease-free survival (DFS) in meningioma patients was detected using RNA-seq data base [28] (Supplementary Fig. 2) which is in line with other cancers [29, 30].

TAMs and GAS6 are expressed by macrophages/microglia in meningioma and schwannoma

TAMs and GAS6 expression by tumour cells and macrophages

Immunohistochemistry demonstrated the expression of pMERTK/MERTK/pAXL/AXL/TYRO3 by tumour cells, macrophages, and endothelial cells in meningioma tissue (Supplementary Fig. 3). Immunofluorescence confirmed the presence of macrophages expressing MERTK/AXL/TYRO3/GAS6 in meningioma and schwannoma tissue. Tumour and other non-macrophage cells (CD68-) also expressed TAMs and GAS6 (Fig. 2A, B).

Fig. 2 — A, B MERTK, AXL, TYRO3 and GAS6 are expressed in CD68-negative tumour cells (white arrows) and CD68-positive macrophages (yellow arrows) in grade-I meningioma and schwannoma tissue. Omission of primary antibody or an appropriate IgG control antibody were used as controls. Images were captured at 20x magnification and scale bars in (A, B) are 50 μm.Western blot data demonstrating correlation between the expression of MERTK, AXL, TYRO3, GAS6, and CD163 and CD68 in meningioma (C, D) and schwannoma (E, F) across successive passages of cell culture (p0-p4). Data in D and F are normalised to a loading control GAPDH and presented as percentage of p0. Spearman’s rank correlation coefficient ‘r’ was calculated to describe the strength and direction of association between CD68 or CD163 and MERTK, AXL, TYRO3 or GAS6.

Characterisation of macrophage subpopulations

Immunocytochemistry (Supplementary Fig. 4A, B, E–H) revealed the presence of M1 (CD68 + /iNOS + ) and M2 (CD68 + /CD163 + ) macrophages, and microglia (CD68 + /TMEM119 + ) in meningioma and schwannoma passage zero (p0) with no significant difference between Merlin-negative and Merlin-positive samples (Supplementary Fig. 4F, H).

In both meningioma and schwannoma p0 cells, a small portion of CD68-negative cells expressing iNOS (CD68-/iNOS + ) and CD163 (CD68-/CD163 + ) were detected. We identified these cells as SSTR2 + /CD163+ and SSTR2 + /iNOS+ meningioma cells, and S100B + /CD163+ and S100B + /iNOS+ schwannoma cells (Supplementary Fig. 4A, B, E–H, I).

Immunofluorescence confirmed the presence of M1 and M2 macrophages in meningioma and schwannoma tissue, and a small number of CD68-/CD163+ and CD68-/iNOS+ cells (Supplementary Fig. 4C, D). In addition, SSTR2 + /CD163+ and SSTR2 + /iNOS+ staining was observed in meningioma (Supplementary Fig. 4C), and S100B + /CD163+ and S100B + /iNOS+ in schwannoma tissue (Supplementary Fig. 4D).

Macrophages contribute to increased global expression of TAMs and GAS6 in meningioma and schwannoma

Expression of MERTK/AXL/TYRO3/GAS6 decreased in tandem with CD163 and CD68, as cells progress from p0 to p4 (Fig. 2C, E). A consistent pattern emerged in which decreasing CD163 expression across successive passages coincided with reduced CD68 expression (Fig. 2C, E). MERTK/TYRO3/GAS6 expression significantly correlate with the decrease of CD163 and CD68 in meningioma and schwannoma cultures (Fig. 2D, F).

AXL expression was only correlated with CD163 expression in meningioma (Fig. 2D), and in schwannoma with both CD68 and CD163 (Fig. 2F). Thus, similarly to tumour cells, macrophages also express MERTK/AXL/TYRO3 and GAS6.

AXL and MERTK promote meningioma and schwannoma proliferation

AXL knockdown (KD) decreased proliferation of meningioma cells, and MERTK-KD decreased proliferation of both meningioma and schwannoma cells detected using Ki67 staining (>p3) (Fig. 3A, F, D, G). This is supported by AXL-KD and MERTK-KD leading to decreased expression of Cyclin D1 (Fig. 3H, I, J). TYRO3-KD had no effect on proliferation in either meningioma or schwannoma (Fig. 3C, F, H, I, E, G, H, J).

Fig. 3 — A–J MERTK depletion using shRNA decreased proliferation of meningioma and schwannoma cells, and AXL depletion decreased proliferation of meningioma cells determined by reduced number of Ki67+ cells and decreased expression of Cyclin D1. TYRO3 depletion had no effect on proliferation in either of tumour cell type. In (F, G) data are normalised to total cell number monitored by DAPI staining and presented as percentage of scramble control (Scr). In (I, J), data are normalised to GAPDH and are presented as percentage of scramble (Scr). One-way ANOVA with post hoc Tukey’s was used with statistical values as follows: ns (not significant) P > 0.05, * P < 0.05, ** P < 0.01. Numbers on the X axis in F, G, I and J graphs refer to different shRNA constructs. In figures the mean ± SEM is given. Scale bars in (A–C), are 40 μm (×20 magnification), in (D) 100 μm (×10 magnification), in (E) 50 μm in the left panel (upper ×10 magnification, and lower ×20 magnification) and 100 μm in the right panel (×10 magnification).

TAMs are inter-dependent in meningioma

MERTK-KD strongly decreased the expression of AXL and TYRO3 in meningioma and schwannoma cells (>p3) (Fig. 4A, D, E). This is in line with MERTK activation known to phosphorylate transcription factor cAMP response element-binding protein (CREB) and regulate the transcription of AXL and TYRO3 by binding to their promoter [31, 32]. AXL-KD reduced the expression of MERTK in meningioma (Fig. 4B, D) but not in schwannoma [26]. In contrast, TYRO3-KD did not decrease AXL or MERTK expression in either tumour type (Fig. 4C–E). Investigating unspecific knockdown, quantitative PCR (qPCR) in meningioma cells demonstrated that two of three MERTK-shRNAs had no effect on either AXL-mRNA or TYRO3-mRNA expression. Additionally, two of three AXL-shRNAs had no effect on either MERTK or TYRO3-mRNA expression (Fig. 4F). Sequence alignment (Supplementary Table 5) demonstrated that due to the presence of many mismatches between shRNAs and target sequences, it is unlikely that these shRNAs would bind non-specifically. The exceptions are shRNA205 (MERTK) and shRNA653 (AXL) (Supplementary Table 5), which display only three mismatches, and therefore could theoretically have off-target effect [33]. qPCR, however, demonstrated that shRNA653 (AXL) decreased only AXL-mRNA (Fig. 4F). Thus, TAMs co-dependency is post-translationally regulated.

TAMs interact/colocalise in schwannoma and meningioma

AXL and MERTK are colocalised at the cell-membrane in a Ben-Men-I/BM-I cells (Fig. 4G). Furthermore, treatment with the MERTK/FLT3 inhibitor, UNC2025 significantly reduced the levels of both MERTK and AXL at the cell membrane, disrupting colocalisation (Fig. 4G).

To investigate this further we performed immunoprecipitation and demonstrated that MERTK/TYRO3 but not MERTK/AXL or TYRO3/AXL, form heterocomplexes in both meningioma and schwannoma tissue (Fig. 4H, I). However, MERTK/TYRO3 heterocomplex was not detected in BM-1 cells (Fig. 4J). Reflecting the result in tissue, MERTK/AXL or TYRO3/AXL heterocomplexes were not detected in BM-1 cells (Fig. 4J). This contrasts with MERTK/AXL colocalisation observed in BM-I cells (Fig. 4G). Since MERTK/TYRO3 heterocomplexes were detected only in tumour tissue and not in cells (>p3), this suggests the involvement of other cell types that are present in tissues.

UNC2025 and BGB324 decrease proliferation and survival of meningioma and schwannoma cells

MERTK/FLT3 inhibitor UNC2025 (Fig. 5A, B) and AXL inhibitor BGB324 (Fig. 5C, D) decreased total number of meningioma and schwannoma cells and increased number of dead cells (Fig. 5A–D) after 72 hrs and 7 days of treatment. In meningioma cells, but not schwannoma cells, the inhibitory effect of both drugs continued after 7 days (Fig. 5A–D), indicating that meningioma cells are more sensitive to both drugs.

This was supported by UNC2025 and BGB324 decreasing the number of proliferating Ki67+ cells in meningioma and schwannoma and increasing the number of apoptotic Cleaved caspase-3+ (C. caspase-3 + ) cells in both tumours (Fig. 5E–L). Both UNC2025 (Fig. 5M, N) and BGB324 (Fig. 5O, P) reduced Cyclin D1 expression in meningioma and schwannoma cells. HMC cells were less sensitive to the inhibitory effect of UNC2025, and equally sensitive to the inhibitory effect of BGB324, on proliferation and survival compared to meningioma cells (Supplementary Fig. 5; Fig. 5A, C).

UNC2025 and BGB324 display differing inhibitory efficacies against TAMs in meningioma and schwannoma

UNC2025

In meningioma cells, UNC2025 decreased pMERTK after 1 hr and 72 hrs and MERTK after 72 hrs of treatment (Fig. 6A, C, D, Supplementary Table 3). UNC2025 non-significantly decreased pTYRO3 and MERTK after 72 hrs but had no effect on pAXL/AXL/TYRO3 at either time points (Supplementary Fig. 6A–D, Supplementary Table 3).

In schwannoma cells, UNC2025 decreased pMERTK after 1 hr and 72 hrs (Fig. 6A, E, Supplementary Table 4). It also decreased pAXL after 1 hr but had no effect on pAXL after 72 hrs (Fig. 6A, F, Supplementary Table 4). There was a non-significant decrease in MERTK/pTYRO3/TYRO3, but not AXL after 72 hrs (Supplementary Fig. 6F–I, Supplementary Table 4).

BGB324

In meningioma cells, BGB324 decreased pAXL after 1 hr and 72 hrs (Fig. 6B, G, Supplementary Table 3). The expression of AXL also decreased after 72 hrs (Fig. 6B, H, Supplementary Table 3). Additionally, BGB324 decreased pMERTK after 1 h and pMERTK/ pTYRO3/TYRO3 after 72 hrs (Fig. 6B, I, J, K, Supplementary Table 3).

In schwannoma cells, BGB324 decreased pAXL only after 72 hrs (Fig. 6B, L), while having no effect on AXL/pTYRO3/TYRO3/pMERTK/MERTK at either time points (Supplementary Fig. 5J–N Supplementary Table 4).

Thus, UNC2025 significantly decreased pMERTK/MERTK/pAXL/AXL in meningioma, but only pMERTK in schwannoma. Meanwhile, BGB324 decreased pMERTK/pAXL/AXL/pTYRO3/TYRO3 in meningioma, and only pAXL in schwannoma.

UNC2025 and BGB324 display different efficacies in the inhibition of signalling pathways in meningioma and schwannoma

ERK/AKT/JNK/FAK/Src are involved in increased proliferation and survival in meningioma and schwannoma [26, 27, 34, 35] and are regulated by AXL in schwannoma [26].

UNC2025

In meningioma cells UNC2025 decreased pERK/pAKT/pJNK after 1 h and 72 hrs (Fig. 7A, B, Supplementary Table 3). UNC2025 also decreased pFAK/pSrc in meningioma cells, but only after 72 hrs (Fig. 7A, B, Supplementary Table 3). UNC2025 did not affect ERK/AKT/JNK/Src, and FAK decreased only after 72 hrs (Fig. 7I, J, Supplementary Table 3).

In schwannoma cells UNC2025 decreased pERK/pAKT/pJNK within 1 hr and 72 hrs (Fig. 7C, D, Supplementary Table 4). No significant decrease was observed in ERK/AKT/JNK/FAK/ Src (Fig. 7K, L, Supplementary Table 4).

BGB324

In meningioma cells BGB324 decreased pERK/pAKT/pJNK/pFAK after 1 hr and 72 hrs (Fig. 7E, F, Supplementary Table 3). pSrc decreased only after 72 hrs (Fig. 7E, F, Supplementary Table 3). BGB324 had no significant effect on ERK/AKT/JNK/FAK/ Src (Fig. 7M, N, Supplementary Table 3).

In schwannoma cells BGB324 decreased pERK/pAKT/pJNK after 1 hr and 72 hrs (Fig. 7G, H, Supplementary Table 4). BGB324 had no significant effect on ERK/AKT/JNK/FAK at either time points. However, it significantly decreased Src after 72 hrs (Fig. 7O, P, Supplementary Table 4).

Thus, UNC2025 decreased pERK/pAKT/pJNK/pFAK/pSrc in meningioma, and pERK/pAKT/pJNK in schwannoma, and BGB324 pERK/ pAKT/pJNK/pFAK/pSrc in both tumour types. UNC2025 was more effective than BGB324 in both tumours (Supplementary Tables 3 and 4).

Tumour cells and macrophages are targetable in meningioma and schwannoma

Although, Cre;Nf2fl/fl schwannoma mouse model [36] recapitulates tumour microenvironment, there are no good genetically engineered meningioma models. Orthotopic xenograft meningioma mice are immunosuppressed and therefore not suitable for tumour microenvironment studies. We have in this study used an in vitro model of patient-derived p0 cell cultures to investigate the targetability of macrophages in meningioma and schwannoma tumours.

Since UNC2025 is more efficacious than BGB324 in decreasing proliferation/survival of both meningioma and schwannoma cells ( > p3), we have tested the effect of this drug in p0 cell cultures in both tumour cell types.

UNC2025 treatment (3 µM, 48 hrs) decreased total macrophages (CD68 +), M1 macrophages (CD68 + /iNOS +), M2 macrophages (CD68 + /CD163 +), and microglia (CD68 +/TMEM119) (Fig. 8A, B, D, E) with no difference observed between Merlin-positive and Merlin-negative tumours. UNC2025 also decreased CD68-/iNOS+ and CD68-/CD163+ non-macrophage cells, including SSTR2 +, SSTR2 + /iNOS+ and SSTR2 + /CD163+ cells in meningioma, and S100B +, S100B +/iNOS+ and S100B +/CD163+ cells in schwannoma. (Fig. 8A, B, D, E).

Fig. 8 — A, B, D, E UNC2025 (3 µM, 48 hrs) decreases CD68-positive macrophage population (CD68 +/CD163+ macrophages, CD68 +/iNOS+ macrophages, and CD68 +/TMEM119+ microglia) in meningioma and schwannoma p0 cells. A decrease of non-macrophagic CD163 +/CD68- and iNOS +/CD68- cell populations, predominantly composed of SSTR2 +, SSTR2 +/iNOS+ and SSTR2 +/CD163+ meningioma cells, and S100B +, S100B +/iNOS+ and S100B +/CD163+ schwannoma cells, is also observed. C, G, F, J UNC2025 (3 µM, 48 hrs) decreases CD68 +/Ki67+ macrophage, TMEM119 +/Ki67+ microglia, and CD68-/Ki67+ non-macrophagic cell populations, including SSTR2 +/Ki67+ meningioma (C, G) and S100B +/Ki67+ schwannoma (F, J) tumour cells. C, H, F, K UNC2025 (3 µM, 48 hrs) increases number of apoptotic CD68 +/Cleaved caspase-3+ macrophages, and CD68-/Cleaved caspase-3+ non-macrophagic cell populations including SSTR2 +/Cleaved caspase-3+ meningioma and S100B +/Cleaved caspase-3+ schwannoma cells. In meningioma (I) but not schwannoma (L) p0 cells, CD68-negative non-macrophage cells (comprising of mostly tumour cells) display a weaker response to UNC2025 (3 µM, 48 hrs) when a higher macrophage population is present. These data are presented as a correlation between macrophage population (CD68+ as a % of total cell number, DAPI) and Ki67 +/CD68- cells responding to the treatment (% of total cell number, DAPI). Unless indicated, images were taken using a Leica SPE confocal microscope at 20x objective magnification. Scale bars are 50 μm. In (A, D), the data are presented as percentage for total cell number (% of DAPI) normalised to EtOH vehicle control. In (G, H, J, K) the data are presented as percentage for total cell number. Statistical tests: (A, D) Single-sample t test or Wilcoxon; (G, H, J, K) two-sample t test or Wilcoxon test; (I) Shapiro–Wilk test, Pearson’s correlation coefficient ‘r’; (L) Shapiro–Wilk test, Spearman’s rank correlation coefficient ‘r’. Statistical values are reported as ns (not significant) P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.

In addition, UNC2025 decreased the number of proliferating macrophages (CD68 +/Ki67 +), which are potentially microglia (TMEM119 +). The proliferation of CD68-negative non-macrophagic cells including tumour cells (SSTR2+ meningioma, S100B+ schwannoma) also decreased (Fig. 8C, F, G, J). UNC2025 treatment weakly increased the number of macrophages expressing Cleaved caspase-3. We observed a similar trend in Cleaved caspase-3 in non-macrophagic cells (CD68-), including tumour cells (Fig. 8C, F, H, K).

We report a moderate negative correlation between a high number of macrophages and a decline in proliferating non-macrophage cells in p0 meningioma cell cultures treated with UNC2025 (Fig. 8I). A similar trend was observed in SSTR2+ meningioma tumour cells (Supplementary Fig. 4J), suggesting that increased macrophage presence reduces the responsiveness of meningioma tumour cells to UNC2025 treatment. No such correlation was observed for p0 schwannoma cell cultures (Fig. 8L).

Discussion

We present MERTK as a new potential therapeutic target in meningioma and schwannoma tumours and propose targeting of MERTK on both tumour cells and macrophages as a new treatment option.

TAMs and GAS6 in tumour cells and tissue

We demonstrate overexpression of pMERTK/MERTK/pAXL/AXL/pTYRO3/TYRO3 in meningioma tissue of all grades compared to NMT. Similarly, to schwannoma [26], GAS6 is overexpressed and released by meningioma tissue and cells. Our RNA-seq analysis suggests that other ligands are also expressed, however, validation using Western blotting is needed. pAXL/AXL/pMERTK/MERTK/pTYRO3 are also overexpressed in schwannoma tissue compared to NNT.

No significant difference was observed between Merlin-positive and Merlin-negative meningioma and schwannoma samples for the activation or expression of any of TAMs or GAS6, with the exception for pTYRO3 being increased in grade-I Merlin-positive meningioma tissue, which has not been confirmed in cells. This is in line with Merlin reintroduction experiments in which expression of AXL/MERTK/TYRO3 in meningioma, and AXL/TYRO3/GAS6 in schwannoma [26] is only partially dependent on Merlin. Expression of MERTK in schwannoma and GAS6 in meningioma is Merlin-independent, suggesting the involvement of other factors.

Increased activation of TAMs in meningioma and schwannoma, can be GAS6-dependent as GAS6 is overexpressed/released by both tumour types. Another MERTK/TYRO3 ligand, GAL3 could also be involved as it is upregulated in meningioma [10], and we uncovered MERTK/TYRO3 heterodimers in both meningioma and schwannoma tissue. Also, activation may occur via ligand-independent heterodimerization within TAM family members [37], or with EGFR/PDGFR/c-MET/HER2 receptors [14], which are all overexpressed in schwannoma and meningioma [27, 38].

TAMs and GAS6 in macrophages

Using immunofluorescence method, we demonstrated that although AXL and MERTK are restricted to anti-inflammatory M2 polarized macrophages in other tumours [18, 39] they are strongly expressed by both M1 and M2 macrophages, and by microglia in meningioma and schwannoma. GAS6 is also expressed by macrophages in meningioma and schwannoma.

These data could not be confirmed by flow cytometry method since anti-AXL and anti-MERTK antibodies, suitable for flow cytometry, did not work in our primary cells.

Characterisation of macrophage subpopulations

We demonstrate the presence of CD68 +/iNOS+ and CD68 +/CD163+ cells in meningioma and schwannoma. While M1 and M2 macrophage have traditionally been marked by iNOS and CD163 respectively, a cytokine profiling should be performed [40].

We exhibited a minor proportion of SSTR2+ meningioma cells and S100B+ schwannoma cell expressing CD163 or iNOS. Our findings align with previous studies indicating the presence of CD163 and iNOS in various cancers [41, 42] including meningioma [43].

We demonstrate the presence of CD68 +/TMEM119+ microglia in meningioma and schwannoma. Self-renewing microglia, normally exclusive to central nervous system [44], are unexpected within peripheral nervous system (PNS) where schwannoma is located. However, a recent study demonstrated that microglia could migrate to the PNS during nerve injury [45]. Microglia is present in meningioma [22].

MERTK/TYRO3 heterodimers formation and a possible role of macrophages

We also identified the endogenous TYRO3/MERTK heterocomplex in meningioma and schwannoma tissue. Although AXL/TYRO3 physical interaction has been reported in other cell types [14, 16], this is the first study to report MERTK/TYRO3 heterocomplex. A specific double band for MERTK is detected in both meningioma and schwannoma cells/tissue. These bands may represent either different isoforms [46] or differently glycosylated MERTK [47]. Anti-TYRO3 antibody pulled down only lower MERTK band in meningioma and only higher MERTK band in schwannoma suggesting that TYRO3 forms complex with different MERTK forms in each tumour type. We demonstrate that MERTK is glycosylated in schwannoma tissue (Supplementary Fig. 1Y).

TYRO3 can form a homo/heterocomplex either in the presence [14] or absence [37] of GAS6. We demonstrated that GAS6 is expressed and released by BM-I cells. However, MERTK/TYRO3 heterodimers have not been detected in these cells suggesting that either GAS6 alone is not sufficient to induce complex formation, or its concentration is insufficient. A study demonstrated that even though GAS6 can induce AXL homodimerization and heterodimerization with MERTK or TYRO3, it alone is unable to induce MERTK or TYRO3 homodimerization/heterodimerisation [48]. This suggests that MERTK/TYRO3 heterodimer formation in schwannoma and meningioma tissue is mediated either by GAS6 acting together with complementary factors or by GAS6-independent mechanisms. Our RNA-seq data reveal expression of GAS6/PROS1/GAL3/TULP1 in meningioma tissue. GAL3 similarly to GAS6 is expressed by macrophages [49]. Since GAL3 is a ligand for both MERTK and TYRO3 [12, 50] it could contribute to MERTK/TYRO3 heterodimerisation, involving both tumour cells and macrophages.

Crosstalk between tumour cells and macrophages is established [51–53] and TAMs expressed and GAS6 expressed/released by both tumour cells and macrophages [18, 51] or microglia [19] can contribute to this crosstalk. Since TAMs can form homo/heterodimers with receptors located on neighbouring cells [14, 16] the MERTK/TYRO3 complex found in meningioma and schwannoma tissue could be a result of such interaction.

Interdependencies between the members of TAM family receptors

MERTK-KD decreased AXL and TYRO3 expression in meningioma and schwannoma cells, while AXL-KD decreased MERTK only in meningioma cells and had no effect on TYRO3 in either tumour type. TYRO3-KD had no effect on MERTK or AXL in either tumour type. Sequence alignment revealed that due to the presence of many mismatches between shRNAs and target sequences, it is unlikely that these shRNAs are unspecific. Moreover, UNC2025 treatment that decreased pMERTK/pAXL after 1hr and MERTK/AXL after 72 hrs in meningioma cells, reduced MERTK/AXL localisation at the cell membrane in BM-I cells, confirming the MERTK/AXL interdependency. In agreement to our results, previous studies demonstrated that lack of MERTK reduced the expression of AXL [15] and TYRO3 [54] in other cell types.

Our qPCR experiments demonstrated that TAM receptor interdependency observed in meningioma and schwannoma is most likely regulated post-translationally.

AXL-shRNA reduced MERTK in meningioma but not in schwannoma cells, and MERTK-shRNA reduced AXL in schwannoma but not in A549 NSCLC cells [31] suggesting that this interdependency is cell-specific.

Although the expression of AXL and TYRO3 strongly depends on MERTK in meningioma and schwannoma cells, no MERTK/AXL and TYRO3/AXL heterodimers were detected in either tumour type. This could be due to the timing of heterodimer formation and/or heterodimer stability [55]. Also, the crystal structure of a GAS6/AXL homodimer demonstrated that GAS6 brings together two AXL monomers forming a homodimer without direct binding between each monomer [56, 57].

Targeting TAMs in tumour cells

MERTK and AXL promote proliferation/survival of meningioma and schwannoma [26] which agrees with studies in other cancers [9, 13, 14]. Although, pTYRO3/TYRO3 was not involved in the increased proliferation or survival in either tumours, its role in increased invasion in higher meningioma grades, or drug resistance in meningioma and schwannoma cannot be excluded [58].

AXL inhibitor BGB324 was more effective in decreasing proliferation/survival in meningioma compared to schwannoma cells, whereas MERTK/FLT3 inhibitor UNC2025 was equally effective in both tumours. The reason for this could be that BGB324 inhibits both pAXL and pMERTK in meningioma cells, and only pAXL in schwannoma. As UNC2025 strongly inhibits pMERTK but not pAXL and is equally effective in decreasing proliferation/survival in both tumour types, this suggests that the inhibition of pMERTK is sufficient for inhibition of proliferation/survival of both tumour types.

We demonstrate that different signalling pathways are regulated by different TAMs in meningioma and schwannoma cells. This could partially explain differences in drug efficacies in these tumours.

Targeting MERTK in macrophages/microglia

We have successfully decreased not only number of tumour cells but also M1/M2 macrophages and microglia in meningioma and schwannoma p0 cells with MERTK/FLT3 inhibitor UNC2025.

The response of p0 meningioma cells to UNC2025 with high macrophage populations was weaker compared to cultures with low macrophage populations. This agrees with studies in other cancers demonstrating reduced sensitivity to drug treatments caused by TAMs expressing macrophages [59]. The decreased sensitivity to UNC2025 in meningioma cells in presence of high macrophage population could be due to macrophage contribution to formation of MERTK/TYRO3 heterocomplexes, as well as elevated concentration of released GAS6, both increasing the levels of active MERTK in meningioma tumour cells [51].

Materials and methods

Cells and tissue

Following informed consent, meningioma and schwannoma samples (Tables S1, S2 containing NF2 status) were obtained pre-op from patients in accordance with ethical approvals (Plymouth Brain Tumour Biobank, REC No: 19/SC/0267, IRAS project ID: 246667). Normal peroneal nerve tissue (NNT) and frozen/paraffin-embedded tissues were obtained from the BRAIN UK biobank (Reference:15/011 and 14/015) [60]. Normal meningeal tissues (NMT) were from Analytical Biological Services Inc. Tumour cells were isolated and cultured as previously described [26, 61]. Human meningeal cells (HMC) (ScienCell Research Laboratories, San Diego, CA, USA) and Merlin-negative meningioma grade-I cell-line (Ben-Men-I/BM-I) (Leibniz Institute DSMZ, Braunschweig, Germany) were cultured as previously described [62].

Genomic analysis

Total genomic DNA was extracted and sequenced or genotyped using KASP^TM as previously described by our group [40, 63].

shRNA knockdown

Lentivirus infections were performed as previously described [61]. Supplementary Table 6 provides a complete list of shRNA lentivirus particles.

RNA isolation and gene expression analysis

Quantitative PCR was conducted as previously described [63] employing the following assays (TFS): MERTK (Hs01031979_m1), TYRO3 (Hs03986773_m1), AXL (Hs01064444_m1). The ΔΔCt method was used to calculate gene expression [64]. All ΔΔCt values were normalised to the housekeeping genes GAPDH and Rpl37a.

Merlin re-introduction

Wild-type-Merlin recombinant adenovirus (AdNF2) and control GFP-containing vector adenoviruses were a gift from J. Testa (Fox Chase Cancer Centre, Philadelphia, PA) [65]. Cells were infected as previously described [34].

Measurement of cell death and total cell number

Cyto Tox-Glo^TM assay (Promega Madison, WI, USA) was performed according to manufacturer’s protocol. Total cell numbers were determined as a percentage of vehicle (EtOH or DMSO). Dead cells were determined as a percentage of total cells then as a percentage of vehicle.

Antibodies

Listed in Supplementary Table 7.

Immunofluorescence

Immunofluorescence was performed as previously described [61, 62]. Additionally, tissues were incubated in 0.3% Sudan Black in 70% EtOH (Thermo Fisher Scientific (TFS), Waltham, MA, USA) for 1 hr at room temperature. Images were taken using 10x, 20x or 40x objectives on a Leica SPE or Leica SP8 microscope and processed with Fiji [66] or LAS-X by Leica (Wetzlar, Germany). Z-stack images were captured for analysis of colocalisation.

Immunohistochemistry

FFPE tissue sections were prepared and stained by the Plymouth Hospital’s neuro-pathology department. Immunohistochemistry was performed as previously described [61]. Images were acquired with the Leica DMRB.

Co-immunoprecipitation

Cells were lysed [61], and frozen tumour pieces were ground in a homogenizer, using ice-cold low salt Triton X-100 lysis buffer (30 mM Tris (pH 8.0), 75 mM NaCl, 10% glycerol and 1% Triton X-100), containing 1% of protease inhibitor (TFS) and 1% of phosphatase inhibitor (Santa Cruz Biotechnology, Dallas, TX, USA). Protein lysates were incubated with protein G Sepharose beads (GE Healthcare, Buckinghamshire, UK) and AXL/MERTK/TYRO3 primary antibodies, at 4 °C overnight. An IgG antibody matched to the species of the antibodies used for immunoprecipitation was used as a negative control.

De-glycosylation

Protein de-glycosylation was performed as previously described [67].

Western blotting

Cell lysis, electrophoresis and Western blotting were performed as previously described [61]. Films were developed using a Compact X4 X-ray film processor (Xograph, Stonehouse, UK), and scanned at a resolution of 600 dpi using a HP Scanjet 2400. Alternatively, membranes were visualised on a PXi Image Analysis System (Syngene, Cambridge, UK). Fiji was used for densitometry quantification [66].

RNA seq and proteomics analysis

Gene expression analysis was performed on the transcriptomic dataset of Van de Weijer et al. [63]. Z-scores of FPKM values were displayed as relative expression between tumour tissue and 2D cultures. For sequence alignment, shRNA target sequences were mapped to reference gene sequences of Tyro3 (gene ID: 7301), Axl (gene ID: 558) and Mertk (gene ID: 10461) using Geneious prime with sensitivity and fine-tuning set to the highest sensitivity and up to five iterations.

Drugs

UNC2025 (Cayman Chemical) (MERTK 0.74 nM; FLT3 0.8 nM, AXL 14 nM; TYRO3 17 nM) [68] and BGB324 (BerGenBio) (AXL 14 nM; MERTK > 50 and TYRO3 > 100fold compared to AXL) [69].

Data Analysis

According to Wu et al. [70] valid application of Western blotting requires starting with three independent biological replicates. This is an accepted standard in the field. Consequently, we adopted this guideline for all our assays. Shapiro–Wilk test, Single-sample Student’s t test, Two-sample Student’s t test, Wilcoxon signed-rank test, one-way ANOVA with post hoc Tukey’s test, or Kruskal Wallis test followed by post hoc Dunn’s test, were used for statistical analysis. Bartlett’s test could not compute variance in data that were normalised to a control. Statistical values are summarised as follows: P > 0.05 or ns (not significant), * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Where relevant, mean ± SEM is indicated. GraphPad Prism (Boston, MA, USA) was used to determine Spearman’s rank and Pearsons’s correlation coefficient and graph data. Microsoft Excel (Redmond, WA, USA) was used to determine IC50 and 95% confidence interval values and graph data. Numbers of biological repeats for each figure are presented in Supplementary Table 8.

Supplementary information

Supplementary table 1^{(50.5KB, xlsx)}

Supplementary table 2^{(21.4KB, xlsx)}

Supplementary figures^{(4.2MB, pdf)}

Acknowledgements

We thank patients, staff at University Hospitals Plymouth, North Bristol NHS trust and the NHS Blood and Transplant community, BRAIN UK, Brain Tumour Research, British Neuropathological Society and Medical Research Council.

Author contributions

FD, KH: investigation/analysis/interpretation/manuscript revision. AL, SH, LH: investigation; LLVDW: investigation/analysis. AS, CLA: investigation/analysis/manuscript revision. EE: cell culture/investigation. DAH: investigation. EAM: sequence alignment. SA: conceptualisation/funding acquisition/investigation/interpretation/original draft writing; COH: conceptualisation/funding acquisition/manuscript revision. KMK: tumour specimen.

Funding

This work was supported by Brain Tumour Research. Kevin Herrera was funded by Animal Free Research UK (Grant number: 083). Animal Free Research UK is the UK’s leading non-animal biomedical research charity that exclusively funds and promotes human relevant research that replaces the use of animals.

Data availability

Supporting data are available in Additional Supplementary Information.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

Participants provided written informed consent and the study was conducted in accordance with the Declaration of Helsinki under institutional review board approval (Plymouth Brain Tumour Biobank, REC No: 19/SC/0267, IRAS project ID: 246667; Reference:15/011 and 14/015).

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Foram Dave, Kevin Herrera, Sylwia Ammoun, C. Oliver Hanemann.

Change history

9/26/2024

A Correction to this paper has been published: 10.1038/s41388-024-03177-z

Contributor Information

Sylwia Ammoun, Email: sylwia.ammoun@plymouth.ac.uk.

C. Oliver Hanemann, Email: oliver.hanemann@plymouth.ac.uk.

Supplementary information

The online version contains supplementary material available at 10.1038/s41388-024-03131-z.

References

1.Plotkin SR, Messiaen L, Legius E, Pancza P, Avery RA, Blakeley JO, et al. Updated diagnostic criteria and nomenclature for neurofibromatosis type 2 and schwannomatosis: An international consensus recommendation. Genet Med. 2022;24:1967–77. [DOI] [PubMed] [Google Scholar]
2.Blakeley JO, Evans DG, Adler J, Brackmann D, Chen R, Ferner RE, et al. Consensus recommendations for current treatments and accelerating clinical trials for patients with neurofibromatosis type 2. Am J Med Genet A. 2012;158A:24–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Agnihotri S, Jalali S, Wilson MR, Danesh A, Li M, Klironomos G, et al. The genomic landscape of schwannoma. Nat Genet. 2016;48:1339–48. [DOI] [PubMed] [Google Scholar]
4.Wang JZ, Nassiri F, Mawrin C, Zadeh G. Genomic Landscape of Meningiomas. Adv Exp Med Biol. 2023;1416:137–58. [DOI] [PubMed] [Google Scholar]
5.Champeaux-Depond C, Weller J, Resche-Rigon M. Neurofibromatosis type 2: a nationwide population-based study focused on survival after meningioma surgery. Clin Neurol Neurosurg. 2020;198:106236. [DOI] [PubMed] [Google Scholar]
6.Jacob JT, Link MJ, Pollock BE. Role of stereotactic radiosurgery in meningiomas and vestibular schwannomas. Curr Treat Options Neurol. 2014;16:308. [DOI] [PubMed] [Google Scholar]
7.Rogers L, Barani I, Chamberlain M, Kaley TJ, McDermott M, Raizer J, et al. Meningiomas: knowledge base, treatment outcomes, and uncertainties. A RANO review. J Neurosurg. 2015;122:4–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Nakatomi H, Jacob JT, Carlson ML, Tanaka S, Tanaka M, Saito N, et al. Long-term risk of recurrence and regrowth after gross-total and subtotal resection of sporadic vestibular schwannoma. J Neurosurg. 2017;133:1052–58. [DOI] [PubMed] [Google Scholar]
9.Vouri M, Hafizi S. TAM receptor tyrosine kinases in cancer drug resistance. Cancer Res. 2017;77:2775–8. [DOI] [PubMed] [Google Scholar]
10.Al Kafri N, Hafizi S. Tumour-secreted protein S (ProS1) activates a Tyro3-Erk signalling axis and protects cancer cells from apoptosis. Cancers (Basel). 2019;11:1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Jung Y, Decker AM, Wang J, Lee E, Kana LA, Yumoto K, et al. Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow. Oncotarget. 2016;7:25698–711. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Caberoy NB, Alvarado G, Bigcas JL, Li W. Galectin-3 is a new MerTK-specific eat-me signal. J Cell Physiol. 2012;227:401–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Vouri M, An Q, Birt M, Pilkington GJ, Hafizi S. Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells. Oncotarget. 2015;6:16183–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Vouri M, Croucher DR, Kennedy SP, An Q, Pilkington GJ, Hafizi S. Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells. Oncogenesis. 2016;5:e266. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Angelillo-Scherrer A, Burnier L, Flores N, Savi P, DeMol M, Schaeffer P, et al. Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy. J Clin Invest. 2005;115:237–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Brown JE, Krodel M, Pazos M, Lai C, Prieto AL. Cross-phosphorylation, signaling and proliferative functions of the Tyro3 and Axl receptors in Rat2 cells. PLoS One. 2012;7:e36800. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Taylor IC, Roy S, Varmus HE. Overexpression of the Sky receptor tyrosine kinase at the cell surface or in the cytoplasm results in ligand-independent activation. Oncogene 1995;11:2619–26. [PubMed] [Google Scholar]
18.Chiu KC, Lee CH, Liu SY, Chou YT, Huang RY, Huang SM, et al. Polarization of tumor-associated macrophages and Gas6/Axl signaling in oral squamous cell carcinoma. Oral Oncol. 2015;51:683–9. [DOI] [PubMed] [Google Scholar]
19.Fourgeaud L, Traves PG, Tufail Y, Leal-Bailey H, Lew ED, Burrola PG, et al. TAM receptors regulate multiple features of microglial physiology. Nature. 2016;532:240–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Giroud P, Renaudineau S, Gudefin L, Calcei A, Menguy T, Rozan C, et al. Expression of TAM-R in human immune cells and unique regulatory function of MerTK in IL-10 production by tolerogenic DC. Front Immunol. 2020;11:564133. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Tanaka M, Siemann DW. Gas6/Axl signaling pathway in the tumor immune microenvironment. Cancers (Basel). 2020;12:1850. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Domingues P, Gonzalez-Tablas M, Otero A, Pascual D, Miranda D, Ruiz L, et al. Tumor infiltrating immune cells in gliomas and meningiomas. Brain Behav Immun. 2016;53:1–15. [DOI] [PubMed] [Google Scholar]
23.Proctor DT, Huang J, Lama S, Albakr A, Van Marle G, Sutherland GR. Tumor-associated macrophage infiltration in meningioma. Neurooncol Adv. 2019;1:vdz018. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Nisenbaum E, Misztal C, Szczupak M, Thielhelm T, Pena S, Mei C, et al. Tumor-associated macrophages in vestibular schwannoma and relationship to hearing. OTO Open. 2021;5:2473974X211059111. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.de Vries M, Briaire-de Bruijn I, Malessy MJ, de Bruine SF, van der Mey AG, Hogendoorn PC. Tumor-associated macrophages are related to volumetric growth of vestibular schwannomas. Otol Neurotol. 2013;34:347–52. [DOI] [PubMed] [Google Scholar]
26.Ammoun S, Provenzano L, Zhou L, Barczyk M, Evans K, Hilton DA, et al. Axl/Gas6/NFkappaB signalling in schwannoma pathological proliferation, adhesion and survival. Oncogene. 2014;33:336–46. [DOI] [PubMed] [Google Scholar]
27.Hilton DA, Shivane A, Kirk L, Bassiri K, Enki DG, Hanemann CO. Activation of multiple growth factor signalling pathways is frequent in meningiomas. Neuropathology. 2016;36:250–61. [DOI] [PubMed] [Google Scholar]
28.Bayley JCT, Hadley CC, Harmanci AO, Harmanci AS, Klisch TJ, Patel AJ. Multiple approaches converge on three biological subtypes of meningioma and extract new insights from published studies. Sci Adv. 2022;8:eabm6247. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Frejno M, Zenezini Chiozzi R, Wilhelm M, Koch H, Zheng R, Klaeger S, et al. Pharmacoproteomic characterisation of human colon and rectal cancer. Mol Syst Biol. 2017;13:951. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Whitman SP, Kohlschmidt J, Maharry K, Volinia S, Mrozek K, Nicolet D, et al. GAS6 expression identifies high-risk adult AML patients: potential implications for therapy. Leukemia. 2014;28:1252–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Linger RM, Cohen RA, Cummings CT, Sather S, Migdall-Wilson J, Middleton DH, et al. Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer. Oncogene. 2013;32:3420–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Kokkinaki M, Abu-Asab M, Gunawardena N, Ahern G, Javidnia M, Young J, et al. Klotho regulates retinal pigment epithelial functions and protects against oxidative stress. J Neurosci. 2013;33:16346–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Amarzguioui M, Holen T, Babaie E, Prydz H. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res. 2003;31:589–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Maze EA, Agit B, Reeves S, Hilton DA, Parkinson DB, Laraba L, et al. Human endogenous retrovirus type k promotes proliferation and confers sensitivity to antiretroviral drugs in merlin-negative schwannoma and meningioma. Cancer Res. 2022;82:235–47. [DOI] [PubMed] [Google Scholar]
35.Suppiah S, Nassiri F, Bi WL, Dunn IF, Hanemann CO, Horbinski CM, et al. Molecular and translational advances in meningiomas. Neuro Oncol. 2019;21:i4–i17. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Gehlhausen JR, Park SJ, Hickox AE, Shew M, Staser K, Rhodes SD, et al. A murine model of neurofibromatosis type 2 that accurately phenocopies human schwannoma formation. Hum Mol Genet. 2015;24:1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Heiring C, Dahlback B, Muller YA. Ligand recognition and homophilic interactions in Tyro3: structural insights into the Axl/Tyro3 receptor tyrosine kinase family. J Biol Chem. 2004;279:6952–8. [DOI] [PubMed] [Google Scholar]
38.Ammoun S, Hanemann CO. Emerging therapeutic targets in schwannomas and other merlin-deficient tumors. Nat Rev Neurol. 2011;7:392–9. [DOI] [PubMed] [Google Scholar]
39.Zizzo G, Hilliard BA, Monestier M, Cohen PL. Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J Immunol. 2012;189:3508–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Adams CL, Ercolano E, Ferluga S, Sofela A, Dave F, Negroni C, et al. A rapid robust method for subgrouping non-NF2 meningiomas according to genotype and detection of lower levels of M2 macrophages in AKT1 E17K mutated tumours. Int J Mol Sci. 2020;21. [DOI] [PMC free article] [PubMed]
41.Vannini F, Kashfi K, Nath N. The dual role of iNOS in cancer. Redox Biol. 2015;6:334–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Ma C, Horlad H, Ohnishi K, Nakagawa T, Yamada S, Kitada S, et al. CD163-positive cancer cells are potentially associated with high malignant potential in clear cell renal cell carcinoma. Med Mol Morphol. 2018;51:13–20. [DOI] [PubMed] [Google Scholar]
43.Kanno H, Nishihara H, Wang L, Yuzawa S, Kobayashi H, Tsuda M, et al. Expression of CD163 prevents apoptosis through the production of granulocyte colony-stimulating factor in meningioma. Neuro Oncol. 2013;15:853–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Davies LC, Jenkins SJ, Allen JE, Taylor PR. Tissue-resident macrophages. Nat Immunol. 2013;14:986–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Green LA, Nebiolo JC, Smith CJ. Microglia exit the CNS in spinal root avulsion. PLoS Biol. 2019;17:e3000159. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Lukovic D, Artero Castro A, Delgado AB, Bernal Mde L, Luna Pelaez N, Diez Lloret A, et al. Human iPSC derived disease model of MERTK-associated retinitis pigmentosa. Sci Rep. 2015;5:12910. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Liu Y, Lan L, Li Y, Lu J, He L, Deng Y, et al. N-glycosylation stabilizes MerTK and promotes hepatocellular carcinoma tumor growth. Redox Biol. 2022;54:102366. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Tsou WI, Nguyen KQ, Calarese DA, Garforth SJ, Antes AL, Smirnov SV, et al. Receptor tyrosine kinases, TYRO3, AXL, and MER, demonstrate distinct patterns and complex regulation of ligand-induced activation. J Biol Chem. 2014;289:25750–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Wang L, Li YS, Yu LG, Zhang XK, Zhao L, Gong FL, et al. Galectin-3 expression and secretion by tumor-associated macrophages in hypoxia promotes breast cancer progression. Biochem Pharm. 2020;178:114113. [DOI] [PubMed] [Google Scholar]
50.Al Kafri N, Hafizi S. Galectin-3 stimulates Tyro3 receptor tyrosine kinase and ERK signalling, cell survival and migration in human cancer cells. Biomolecules. 2020;10. [DOI] [PMC free article] [PubMed]
51.Loges S, Schmidt T, Tjwa M, van Geyte K, Lievens D, Lutgens E, et al. Malignant cells fuel tumor growth by educating infiltrating leukocytes to produce the mitogen Gas6. Blood. 2010;115:2264–73. [DOI] [PubMed] [Google Scholar]
52.Hinshaw DC, Shevde LA. THE TUMOR MICROENVIRONMENT INNATELY MODULATES CANCER PRogression. Cancer Res. 2019;79:4557–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Arneth B. Tumor microenvironment. Medicina (Kaunas). 2019;56. [DOI] [PMC free article] [PubMed]
54.Prasad D, Rothlin CV, Burrola P, Burstyn-Cohen T, Lu Q, Garcia de Frutos P, et al. TAM receptor function in the retinal pigment epithelium. Mol Cell Neurosci. 2006;33:96–108. [DOI] [PubMed] [Google Scholar]
55.Nooren IM, Thornton JM. Diversity of protein-protein interactions. EMBO J. 2003;22:3486–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2010;141:1117–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Sasaki T, Knyazev PG, Clout NJ, Cheburkin Y, Gohring W, Ullrich A, et al. Structural basis for Gas6-Axl signalling. EMBO J. 2006;25:80–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Chien CW, Hou PC, Wu HC, Chang YL, Lin SC, Lin SC, et al. Targeting TYRO3 inhibits epithelial-mesenchymal transition and increases drug sensitivity in colon cancer. Oncogene. 2016;35:5872–81. [DOI] [PubMed] [Google Scholar]
59.Myers KV, Amend SR, Pienta KJ. Targeting Tyro3, Axl and MerTK (TAM receptors): implications for macrophages in the tumor microenvironment. Mol Cancer. 2019;18:94. [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Nicoll JAR, Bloom T, Clarke A, Boche D, Hilton D. BRAIN UK: accessing NHS tissue archives for neuroscience research. Neuropathol Appl Neurobiol. 2022;48:e12766. [DOI] [PubMed] [Google Scholar]
61.Lyons Rimmer J, Ercolano E, Baiz D, Makhija M, Berger A, Sells T, et al. The potential of MLN3651 in combination with selumetinib as a treatment for merlin-deficient meningioma. Cancers (Basel). 2020;12:1744. [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Laraba L, Hillson L, de Guibert JG, Hewitt A, Jaques MR, Tang TT, et al. Inhibition of YAP/TAZ-driven TEAD activity prevents growth of NF2-null schwannoma and meningioma. Brain. 2023;146:1697–713. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.van de Weijer LL, Ercolano E, Zhang T, Shah M, Banton MC, Na J, et al. A novel patient-derived meningioma spheroid model as a tool to study and treat epithelial-to-mesenchymal transition (EMT) in meningiomas. Acta Neuropathol Commun. 2023;11:198. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods. 2001;25:402–8. [DOI] [PubMed] [Google Scholar]
65.Xiao GH, Gallagher R, Shetler J, Skele K, Altomare DA, Pestell RG, et al. The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression. Mol Cell Biol. 2005;25:2384–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Provenzano L, Ryan Y, Hilton DA, Lyons-Rimmer J, Dave F, Maze EA, et al. Cellular prion protein (PrP(C)) in the development of Merlin-deficient tumours. Oncogene. 2017;36:6132–42. [DOI] [PubMed] [Google Scholar]
68.Zhang W, DeRyckere D, Hunter D, Liu J, Stashko MA, Minson KA, et al. UNC2025, a potent and orally bioavailable MER/FLT3 dual inhibitor. J Med Chem. 2014;57:7031–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W, et al. R428, a selective small molecule inhibitor of Axl kinase, blocks tumor spread and prolongs survival in models of metastatic breast cancer. Cancer Res. 2010;70:1544–54. [DOI] [PubMed] [Google Scholar]
70.Wu L, Hu X, Tang H, Han Z, Chen Y. Valid application of western blotting. Mol Biol Rep. 2014;41:3517–20. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary table 1^{(50.5KB, xlsx)}

Supplementary table 2^{(21.4KB, xlsx)}

Supplementary figures^{(4.2MB, pdf)}

Data Availability Statement

Supporting data are available in Additional Supplementary Information.

[CR1] 1.Plotkin SR, Messiaen L, Legius E, Pancza P, Avery RA, Blakeley JO, et al. Updated diagnostic criteria and nomenclature for neurofibromatosis type 2 and schwannomatosis: An international consensus recommendation. Genet Med. 2022;24:1967–77. [DOI] [PubMed] [Google Scholar]

[CR2] 2.Blakeley JO, Evans DG, Adler J, Brackmann D, Chen R, Ferner RE, et al. Consensus recommendations for current treatments and accelerating clinical trials for patients with neurofibromatosis type 2. Am J Med Genet A. 2012;158A:24–41. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Agnihotri S, Jalali S, Wilson MR, Danesh A, Li M, Klironomos G, et al. The genomic landscape of schwannoma. Nat Genet. 2016;48:1339–48. [DOI] [PubMed] [Google Scholar]

[CR4] 4.Wang JZ, Nassiri F, Mawrin C, Zadeh G. Genomic Landscape of Meningiomas. Adv Exp Med Biol. 2023;1416:137–58. [DOI] [PubMed] [Google Scholar]

[CR5] 5.Champeaux-Depond C, Weller J, Resche-Rigon M. Neurofibromatosis type 2: a nationwide population-based study focused on survival after meningioma surgery. Clin Neurol Neurosurg. 2020;198:106236. [DOI] [PubMed] [Google Scholar]

[CR6] 6.Jacob JT, Link MJ, Pollock BE. Role of stereotactic radiosurgery in meningiomas and vestibular schwannomas. Curr Treat Options Neurol. 2014;16:308. [DOI] [PubMed] [Google Scholar]

[CR7] 7.Rogers L, Barani I, Chamberlain M, Kaley TJ, McDermott M, Raizer J, et al. Meningiomas: knowledge base, treatment outcomes, and uncertainties. A RANO review. J Neurosurg. 2015;122:4–23. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR8] 8.Nakatomi H, Jacob JT, Carlson ML, Tanaka S, Tanaka M, Saito N, et al. Long-term risk of recurrence and regrowth after gross-total and subtotal resection of sporadic vestibular schwannoma. J Neurosurg. 2017;133:1052–58. [DOI] [PubMed] [Google Scholar]

[CR9] 9.Vouri M, Hafizi S. TAM receptor tyrosine kinases in cancer drug resistance. Cancer Res. 2017;77:2775–8. [DOI] [PubMed] [Google Scholar]

[CR10] 10.Al Kafri N, Hafizi S. Tumour-secreted protein S (ProS1) activates a Tyro3-Erk signalling axis and protects cancer cells from apoptosis. Cancers (Basel). 2019;11:1843. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR11] 11.Jung Y, Decker AM, Wang J, Lee E, Kana LA, Yumoto K, et al. Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow. Oncotarget. 2016;7:25698–711. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Caberoy NB, Alvarado G, Bigcas JL, Li W. Galectin-3 is a new MerTK-specific eat-me signal. J Cell Physiol. 2012;227:401–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Vouri M, An Q, Birt M, Pilkington GJ, Hafizi S. Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells. Oncotarget. 2015;6:16183–97. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Vouri M, Croucher DR, Kennedy SP, An Q, Pilkington GJ, Hafizi S. Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells. Oncogenesis. 2016;5:e266. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Angelillo-Scherrer A, Burnier L, Flores N, Savi P, DeMol M, Schaeffer P, et al. Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy. J Clin Invest. 2005;115:237–46. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Brown JE, Krodel M, Pazos M, Lai C, Prieto AL. Cross-phosphorylation, signaling and proliferative functions of the Tyro3 and Axl receptors in Rat2 cells. PLoS One. 2012;7:e36800. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Taylor IC, Roy S, Varmus HE. Overexpression of the Sky receptor tyrosine kinase at the cell surface or in the cytoplasm results in ligand-independent activation. Oncogene 1995;11:2619–26. [PubMed] [Google Scholar]

[CR18] 18.Chiu KC, Lee CH, Liu SY, Chou YT, Huang RY, Huang SM, et al. Polarization of tumor-associated macrophages and Gas6/Axl signaling in oral squamous cell carcinoma. Oral Oncol. 2015;51:683–9. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Fourgeaud L, Traves PG, Tufail Y, Leal-Bailey H, Lew ED, Burrola PG, et al. TAM receptors regulate multiple features of microglial physiology. Nature. 2016;532:240–4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Giroud P, Renaudineau S, Gudefin L, Calcei A, Menguy T, Rozan C, et al. Expression of TAM-R in human immune cells and unique regulatory function of MerTK in IL-10 production by tolerogenic DC. Front Immunol. 2020;11:564133. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Tanaka M, Siemann DW. Gas6/Axl signaling pathway in the tumor immune microenvironment. Cancers (Basel). 2020;12:1850. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Domingues P, Gonzalez-Tablas M, Otero A, Pascual D, Miranda D, Ruiz L, et al. Tumor infiltrating immune cells in gliomas and meningiomas. Brain Behav Immun. 2016;53:1–15. [DOI] [PubMed] [Google Scholar]

[CR23] 23.Proctor DT, Huang J, Lama S, Albakr A, Van Marle G, Sutherland GR. Tumor-associated macrophage infiltration in meningioma. Neurooncol Adv. 2019;1:vdz018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Nisenbaum E, Misztal C, Szczupak M, Thielhelm T, Pena S, Mei C, et al. Tumor-associated macrophages in vestibular schwannoma and relationship to hearing. OTO Open. 2021;5:2473974X211059111. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.de Vries M, Briaire-de Bruijn I, Malessy MJ, de Bruine SF, van der Mey AG, Hogendoorn PC. Tumor-associated macrophages are related to volumetric growth of vestibular schwannomas. Otol Neurotol. 2013;34:347–52. [DOI] [PubMed] [Google Scholar]

[CR26] 26.Ammoun S, Provenzano L, Zhou L, Barczyk M, Evans K, Hilton DA, et al. Axl/Gas6/NFkappaB signalling in schwannoma pathological proliferation, adhesion and survival. Oncogene. 2014;33:336–46. [DOI] [PubMed] [Google Scholar]

[CR27] 27.Hilton DA, Shivane A, Kirk L, Bassiri K, Enki DG, Hanemann CO. Activation of multiple growth factor signalling pathways is frequent in meningiomas. Neuropathology. 2016;36:250–61. [DOI] [PubMed] [Google Scholar]

[CR28] 28.Bayley JCT, Hadley CC, Harmanci AO, Harmanci AS, Klisch TJ, Patel AJ. Multiple approaches converge on three biological subtypes of meningioma and extract new insights from published studies. Sci Adv. 2022;8:eabm6247. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Frejno M, Zenezini Chiozzi R, Wilhelm M, Koch H, Zheng R, Klaeger S, et al. Pharmacoproteomic characterisation of human colon and rectal cancer. Mol Syst Biol. 2017;13:951. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Whitman SP, Kohlschmidt J, Maharry K, Volinia S, Mrozek K, Nicolet D, et al. GAS6 expression identifies high-risk adult AML patients: potential implications for therapy. Leukemia. 2014;28:1252–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR31] 31.Linger RM, Cohen RA, Cummings CT, Sather S, Migdall-Wilson J, Middleton DH, et al. Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer. Oncogene. 2013;32:3420–31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Kokkinaki M, Abu-Asab M, Gunawardena N, Ahern G, Javidnia M, Young J, et al. Klotho regulates retinal pigment epithelial functions and protects against oxidative stress. J Neurosci. 2013;33:16346–59. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Amarzguioui M, Holen T, Babaie E, Prydz H. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res. 2003;31:589–95. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Maze EA, Agit B, Reeves S, Hilton DA, Parkinson DB, Laraba L, et al. Human endogenous retrovirus type k promotes proliferation and confers sensitivity to antiretroviral drugs in merlin-negative schwannoma and meningioma. Cancer Res. 2022;82:235–47. [DOI] [PubMed] [Google Scholar]

[CR35] 35.Suppiah S, Nassiri F, Bi WL, Dunn IF, Hanemann CO, Horbinski CM, et al. Molecular and translational advances in meningiomas. Neuro Oncol. 2019;21:i4–i17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Gehlhausen JR, Park SJ, Hickox AE, Shew M, Staser K, Rhodes SD, et al. A murine model of neurofibromatosis type 2 that accurately phenocopies human schwannoma formation. Hum Mol Genet. 2015;24:1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Heiring C, Dahlback B, Muller YA. Ligand recognition and homophilic interactions in Tyro3: structural insights into the Axl/Tyro3 receptor tyrosine kinase family. J Biol Chem. 2004;279:6952–8. [DOI] [PubMed] [Google Scholar]

[CR38] 38.Ammoun S, Hanemann CO. Emerging therapeutic targets in schwannomas and other merlin-deficient tumors. Nat Rev Neurol. 2011;7:392–9. [DOI] [PubMed] [Google Scholar]

[CR39] 39.Zizzo G, Hilliard BA, Monestier M, Cohen PL. Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J Immunol. 2012;189:3508–20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Adams CL, Ercolano E, Ferluga S, Sofela A, Dave F, Negroni C, et al. A rapid robust method for subgrouping non-NF2 meningiomas according to genotype and detection of lower levels of M2 macrophages in AKT1 E17K mutated tumours. Int J Mol Sci. 2020;21. [DOI] [PMC free article] [PubMed]

[CR41] 41.Vannini F, Kashfi K, Nath N. The dual role of iNOS in cancer. Redox Biol. 2015;6:334–43. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR42] 42.Ma C, Horlad H, Ohnishi K, Nakagawa T, Yamada S, Kitada S, et al. CD163-positive cancer cells are potentially associated with high malignant potential in clear cell renal cell carcinoma. Med Mol Morphol. 2018;51:13–20. [DOI] [PubMed] [Google Scholar]

[CR43] 43.Kanno H, Nishihara H, Wang L, Yuzawa S, Kobayashi H, Tsuda M, et al. Expression of CD163 prevents apoptosis through the production of granulocyte colony-stimulating factor in meningioma. Neuro Oncol. 2013;15:853–64. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Davies LC, Jenkins SJ, Allen JE, Taylor PR. Tissue-resident macrophages. Nat Immunol. 2013;14:986–95. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Green LA, Nebiolo JC, Smith CJ. Microglia exit the CNS in spinal root avulsion. PLoS Biol. 2019;17:e3000159. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Lukovic D, Artero Castro A, Delgado AB, Bernal Mde L, Luna Pelaez N, Diez Lloret A, et al. Human iPSC derived disease model of MERTK-associated retinitis pigmentosa. Sci Rep. 2015;5:12910. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR47] 47.Liu Y, Lan L, Li Y, Lu J, He L, Deng Y, et al. N-glycosylation stabilizes MerTK and promotes hepatocellular carcinoma tumor growth. Redox Biol. 2022;54:102366. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR48] 48.Tsou WI, Nguyen KQ, Calarese DA, Garforth SJ, Antes AL, Smirnov SV, et al. Receptor tyrosine kinases, TYRO3, AXL, and MER, demonstrate distinct patterns and complex regulation of ligand-induced activation. J Biol Chem. 2014;289:25750–63. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR49] 49.Wang L, Li YS, Yu LG, Zhang XK, Zhao L, Gong FL, et al. Galectin-3 expression and secretion by tumor-associated macrophages in hypoxia promotes breast cancer progression. Biochem Pharm. 2020;178:114113. [DOI] [PubMed] [Google Scholar]

[CR50] 50.Al Kafri N, Hafizi S. Galectin-3 stimulates Tyro3 receptor tyrosine kinase and ERK signalling, cell survival and migration in human cancer cells. Biomolecules. 2020;10. [DOI] [PMC free article] [PubMed]

[CR51] 51.Loges S, Schmidt T, Tjwa M, van Geyte K, Lievens D, Lutgens E, et al. Malignant cells fuel tumor growth by educating infiltrating leukocytes to produce the mitogen Gas6. Blood. 2010;115:2264–73. [DOI] [PubMed] [Google Scholar]

[CR52] 52.Hinshaw DC, Shevde LA. THE TUMOR MICROENVIRONMENT INNATELY MODULATES CANCER PRogression. Cancer Res. 2019;79:4557–66. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR53] 53.Arneth B. Tumor microenvironment. Medicina (Kaunas). 2019;56. [DOI] [PMC free article] [PubMed]

[CR54] 54.Prasad D, Rothlin CV, Burrola P, Burstyn-Cohen T, Lu Q, Garcia de Frutos P, et al. TAM receptor function in the retinal pigment epithelium. Mol Cell Neurosci. 2006;33:96–108. [DOI] [PubMed] [Google Scholar]

[CR55] 55.Nooren IM, Thornton JM. Diversity of protein-protein interactions. EMBO J. 2003;22:3486–92. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR56] 56.Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2010;141:1117–34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR57] 57.Sasaki T, Knyazev PG, Clout NJ, Cheburkin Y, Gohring W, Ullrich A, et al. Structural basis for Gas6-Axl signalling. EMBO J. 2006;25:80–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR58] 58.Chien CW, Hou PC, Wu HC, Chang YL, Lin SC, Lin SC, et al. Targeting TYRO3 inhibits epithelial-mesenchymal transition and increases drug sensitivity in colon cancer. Oncogene. 2016;35:5872–81. [DOI] [PubMed] [Google Scholar]

[CR59] 59.Myers KV, Amend SR, Pienta KJ. Targeting Tyro3, Axl and MerTK (TAM receptors): implications for macrophages in the tumor microenvironment. Mol Cancer. 2019;18:94. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR60] 60.Nicoll JAR, Bloom T, Clarke A, Boche D, Hilton D. BRAIN UK: accessing NHS tissue archives for neuroscience research. Neuropathol Appl Neurobiol. 2022;48:e12766. [DOI] [PubMed] [Google Scholar]

[CR61] 61.Lyons Rimmer J, Ercolano E, Baiz D, Makhija M, Berger A, Sells T, et al. The potential of MLN3651 in combination with selumetinib as a treatment for merlin-deficient meningioma. Cancers (Basel). 2020;12:1744. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR62] 62.Laraba L, Hillson L, de Guibert JG, Hewitt A, Jaques MR, Tang TT, et al. Inhibition of YAP/TAZ-driven TEAD activity prevents growth of NF2-null schwannoma and meningioma. Brain. 2023;146:1697–713. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR63] 63.van de Weijer LL, Ercolano E, Zhang T, Shah M, Banton MC, Na J, et al. A novel patient-derived meningioma spheroid model as a tool to study and treat epithelial-to-mesenchymal transition (EMT) in meningiomas. Acta Neuropathol Commun. 2023;11:198. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR64] 64.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Methods. 2001;25:402–8. [DOI] [PubMed] [Google Scholar]

[CR65] 65.Xiao GH, Gallagher R, Shetler J, Skele K, Altomare DA, Pestell RG, et al. The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression. Mol Cell Biol. 2005;25:2384–94. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR66] 66.Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR67] 67.Provenzano L, Ryan Y, Hilton DA, Lyons-Rimmer J, Dave F, Maze EA, et al. Cellular prion protein (PrP(C)) in the development of Merlin-deficient tumours. Oncogene. 2017;36:6132–42. [DOI] [PubMed] [Google Scholar]

[CR68] 68.Zhang W, DeRyckere D, Hunter D, Liu J, Stashko MA, Minson KA, et al. UNC2025, a potent and orally bioavailable MER/FLT3 dual inhibitor. J Med Chem. 2014;57:7031–41. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR69] 69.Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W, et al. R428, a selective small molecule inhibitor of Axl kinase, blocks tumor spread and prolongs survival in models of metastatic breast cancer. Cancer Res. 2010;70:1544–54. [DOI] [PubMed] [Google Scholar]

[CR70] 70.Wu L, Hu X, Tang H, Han Z, Chen Y. Valid application of western blotting. Mol Biol Rep. 2014;41:3517–20. [DOI] [PubMed] [Google Scholar]

PERMALINK

Targeting MERTK on tumour cells and macrophages: a potential intervention for sporadic and NF2-related meningioma and schwannoma tumours

Foram Dave

Kevin Herrera

Alex Lockley

Laurien L van de Weijer

Summer Henderson

Agbolahan A Sofela

Laura Hook

Claire L Adams

Emanuela Ercolano

David A Hilton

Emmanuel A Maze

Kathreena M Kurian

Sylwia Ammoun

C Oliver Hanemann

Abstract

Introduction

Results

Expression of TAMs and GAS6 in meningioma and schwannoma

Fig. 1. MERTK/pMERTK, AXL/pAXL, TYRO3/pTYRO3 and GAS6 are overexpressed in meningioma tissue of all grades.

TAMs and GAS6 are expressed by macrophages/microglia in meningioma and schwannoma

TAMs and GAS6 expression by tumour cells and macrophages

Fig. 2. TAM receptors and GAS6 are expressed by tumour-associated macrophages in meningioma and schwannoma tumours.

Characterisation of macrophage subpopulations

Macrophages contribute to increased global expression of TAMs and GAS6 in meningioma and schwannoma

AXL and MERTK promote meningioma and schwannoma proliferation

Fig. 3. AXL and MERTK are involved in the increased proliferation of meningioma and schwannoma cells.

TAMs are inter-dependent in meningioma

Fig. 4. TAM receptors are interdependent in meningioma and schwannoma.

TAMs interact/colocalise in schwannoma and meningioma

UNC2025 and BGB324 decrease proliferation and survival of meningioma and schwannoma cells

Fig. 5. Inhibition of MERTK and AXL decrease proliferation and increase apoptosis in meningioma and schwannoma cells.

UNC2025 and BGB324 display differing inhibitory efficacies against TAMs in meningioma and schwannoma

UNC2025

Fig. 6. UNC2025 and BGB324 inhibit the expression/activation of TAM receptors in meningioma and schwannoma cells.

BGB324

UNC2025 and BGB324 display different efficacies in the inhibition of signalling pathways in meningioma and schwannoma

UNC2025

Fig. 7. UNC2025 and BGB324 inhibit signalling pathways in meningioma and schwannoma cells.

BGB324

Tumour cells and macrophages are targetable in meningioma and schwannoma

Fig. 8. UNC2025 decreases number of macrophages, microglia, and tumour cells in meningioma and schwannoma passage 0 (p0) cells.

Discussion

TAMs and GAS6 in tumour cells and tissue

TAMs and GAS6 in macrophages

Characterisation of macrophage subpopulations

MERTK/TYRO3 heterodimers formation and a possible role of macrophages

Interdependencies between the members of TAM family receptors

Targeting TAMs in tumour cells

Targeting MERTK in macrophages/microglia

Materials and methods

Cells and tissue

Genomic analysis

shRNA knockdown

RNA isolation and gene expression analysis

Merlin re-introduction

Measurement of cell death and total cell number

Antibodies

Immunofluorescence

Immunohistochemistry

Co-immunoprecipitation

De-glycosylation

Western blotting

RNA seq and proteomics analysis

Drugs

Data Analysis

Supplementary information

Acknowledgements

Author contributions

Funding

Data availability

Competing interests

Ethics approval and consent to participate

Footnotes

Contributor Information

Supplementary information

References

Associated Data

Supplementary Materials